Methods for the synthesis of activated ethylfumarates and their use as intermediates

ABSTRACT

Disclosed embodiments relate to improved methods for the synthesis of activated fumarate intermediates and their use in chemical synthesis. Disclosed embodiments describe the synthesis of activated fumarate esters including those derived from activating groups including: 4-nitrophenyl, diphenylphophoryl azide, pivaloyl chloride, chlorosulfonyl isocyanate, p-nitrophenol, MEF, trifluoroacetyl and chlorine, for example, ethyl fumaroyl chloride and the subsequent use of the activated ester in situ. Further embodiments describe the improved synthesis of substituted aminoalkyl-diketopiperazines from unisolated and unpurified intermediates allowing for improved yields and reactor throughput.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional patent application claims the benefit of U.S.non-provisional application Ser. No. 13/834,106, filed Mar. 15, 2013,which in-turn claims the benefit of U.S. provisional application No.61/639,536, filed Apr. 27, 2012, now expired, the content of which arehereby incorporated by reference as if recited herein in their entirety.

TECHNICAL FIELD

The present invention relates to compositions for delivering activeagents, and particularly biologically active agents. Disclosedembodiments are in the field of chemical synthesis and more particularlyare related to improved synthetic methods for the preparation ofethyl-4-nitrophenylfumarate and its use as a chemical intermediate.

BACKGROUND

Drug delivery is a persistent problem in the administration of activeagents to patients. Conventional means for delivering active agents areoften severely limited by biological, chemical, and physical barriers.Typically, these barriers are imposed by the environment through whichdelivery occurs, the environment of the target for delivery, or thetarget itself.

Biologically active agents are particularly vulnerable to such barriers.For example in the delivery to humans of pharmacological and therapeuticagents, barriers are imposed by the body. Examples of physical barriersare the skin and various organ membranes that must be traversed beforereaching a target. Chemical barriers include, but are not limited to, pHvariations, lipid bi-layers, and degrading enzymes.

These barriers are of particular significance in the design of oraldelivery systems. Oral delivery of many biologically active agents wouldbe the route of choice for administration to animals if not forbiological, chemical, and physical barriers such as varying pH in thegastrointestinal (GI) tract, powerful digestive enzymes, and activeagent impermeable gastrointestinal membranes. Among the numerous agentswhich are not typically amenable to oral administration are biologicallyactive peptides, such as calcitonin and insulin; polysaccharides, and inparticular mucopolysaccharides including, but not limited to, heparin;heparinoids; antibiotics; and other organic substances. These agents arerapidly rendered ineffective or are destroyed in the gastrointestinaltract by acid hydrolysis, enzymes, or the like.

Earlier methods for orally administering vulnerable pharmacologicalagents have relied on the co-administration of adjuvants (e.g.,resorcinols and non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation.

Liposomes have also been described as drug delivery systems for insulinand heparin. See, for example, U.S. Pat. No. 4,239,754; Patel et al.(1976), FEBS Letters, Vol. 62, pg. 60; and Hashimoto et al. (1979),Endocrinology Japan, Vol. 26, pg. 337.

However, broad spectrum use of drug delivery systems is precludedbecause: (1) the systems require toxic amounts of adjuvants orinhibitors; (2) suitable low molecular weight cargos, i.e. activeagents, are not available; (3) the systems exhibit poor stability andinadequate shelf life; (4) the systems are difficult to manufacture; (5)the systems fail to protect the active agent (cargo); (6) the systemsadversely alter the active agent; or (7) the systems fail to allow orpromote absorption of the active agent.

More recently, microspheres of artificial polymers of mixed amino acids(proteinoids) have been used to deliver pharmaceuticals. For example,U.S. Pat. No. 4,925,673 describes drug-containing proteinoid microspherecarriers as well as methods for their preparation and use. Theseproteinoid microspheres are useful for the delivery of a number ofactive agents.

There is still a need in the art for simple, inexpensive deliverysystems which are easily prepared and which can deliver a broad range ofactive agents. One class of delivery system that has shown promise isdiketopiperazines (DKP). In particular,3,6-bis-substituted-2,5-diketopiperazines have been shown to effectivelydeliver biologically active agents to the systemic circulation of thelung.

Depending on the DKP and the route of administration, the DKP moleculecan require substitution and/or modification of the side chains attachedto the diketopiperazine ring to optimize the profile of the excipientfor the delivery route at hand. One such group is includesdiketopiperazines with a substituted amino alkyl group, or so-called3,6-aminoalkyl-2,5-diketopiperazines. Substitution of the side-chainamino group often involves reaction with an electrophile. Many factorsenter into the choice of an appropriate electrophile, such as commercialavailability, whether it is appropriate for large scale production or isdifficult to isolate for subsequent reaction with theaminoalkyldiketopiperazine.

The introduction of a fumaroyl side chain onto, for example, a3,6-aminoalkyl-2,5-diketopiperazine has proven especially advantageousas an excipient. However, the introduction of this fumaroyl moietyrequires significant synthetic effort. One option for functionalizationof the DKP utilizes the fact that the aminoalkyl groups may be used asnucleophiles in order to further modify the diketopiperazine excipients.Ethylfumaryl chloride (EFC) is known and available commercially,however, there are disadvantages to pharmaceutical scale use of the acidchloride. Some of the disadvantages, include, limited reactivity,purity, potential for backlogs in commercial availability etc.Therefore, it may be advantageous to increase the reactivity of theelectrophilic site. One way to accomplish this is through thep-nitrophenol ester of ethyl fumarate, ethyl-4-nitrophenylfumarate orother activated ethyl fumarates.

Moreover, there are considerable costs and time pressures involved withany production scale chemical manufacturing endeavor, including that ofexcipients like the aforementioned diketopiperazines. Therefore, thereis a need not only for excipients with optimal physico-chemicalproperties, but also for optimized production scale manufacturing ofthose chemicals. This must take into account not only raw material andreaction costs, but also reactor throughput and time expended insynthesizing the target molecule. The general approach for maximizingoverall yield for a chemical process involves maximizing the yield andpurity of each intermediate along the chemical pathway. This regularlysuggests isolating and purifying each intermediate prior to subsequentreaction. By taking this approach the hope is that: a) by-products andunreacted starting materials from each step are prevented frominteracting with later introduced intermediates or starting materials;and b) purification of the end target is simplified by having previouslyremoved prior by-products, starting materials, etc. and therebymaximizing yield of the end target by reducing the amount of loss due topurification that could take place.

SUMMARY

This and other unmet needs of the prior art are met by compounds andmethods as described in more detail below. The use of substituted3,6-aminoalkyl-2,5-diketopiperazines as pharmaceutical excipients hasshown considerable promise. Of particular interest are carboxysubstituted aminoalkyl-diketopiperazines such as those described byFormula I (R₁═R₂═COOR₃).

Synthesis of carboxy substituted aminoalkyl-diketopiperazines(R₁═R₂═RCOOH) may proceed through an isolatedaminoalkyl-diketopiperazine (for example the compound of the Formula 2)or an acid salt thereof (such as Formula 2). The amine is then reactedwith an appropriate electrophile (for exampleethyl-4-nitrophenylfumarate, 3) to give a substitutedaminoalkyl-diketopiperazine (such as compound 4 R=Et) which, dependingon the target molecule, may then undergo further functionalization orremoval of protecting groups to give substitutedaminoalkyl-diketopiperazines (such as compound 4 R═H).

Generally, the aim of optimizing overall yield in a multi-step chemicalsynthesis is accomplished by isolation and purification of eachintermediate molecule prior to subsequent reaction. This approach hopesto avoid loss of the final target due to: a) by-products of the previoussteps reacting with intermediates or starting materials; and b) loss dueto more complicated isolation and purification of the target molecule.

Disclosed embodiments provide methods for the synthesis of substituteddiketopiperazine pharmaceutical excipients via use of in situ generatedintermediates. The embodiments provide results which, counter to generalthought, achieve higher yield and reactor throughput than traditional,isolate-and-purify-type methods. More specifically, embodiments showmethods for the generation and use of fumaroyl intermediates in situ andwithout purification, as well as methods for the generation and use ofaminoalkyl-diketopiperazines in situ and without isolation orpurification.

In an embodiment, methods for the preparation of activated esters ofmono-ethyl fumarate (MEF) are disclosed. Other embodiments relate to thegeneration of anhydrides of MEF and their use as intermediates. Furtherembodiments relate to the preparation and in situ use of activatedesters of MEF. Further embodiments relate to the generation ofethyl-4-nitrophenylfumarate via the generation of a reactive salt of4-nitrophenol. Further embodiments relate to the generation of activatedesters of MEF from activated 4-nitrophenylesters. In an embodiment, anactivating group, agent or reactant can be selected from a number ofreactants, including, but not limited to diphenylphophoryl azide,pivaloyl chloride, chlorosulfonyl isocyanate, p-nitrophenol, MEF,trifluoroacetyl and chlorine, for example, ethyl fumaroyl chloride.

Disclosed embodiments include a method for the synthesis of an activatedester of MEF comprising: providing a reactive electrophilic derivativeof MEF; reacting an alcohol with an appropriate base and generating asalt of the alcohol, the base chosen from the group comprising: organicand inorganic metallic bases; and reacting the fumaric acid derivativewith the sodium salt in an appropriate solvent. Further embodimentsinclude methods where: the alcohol is 4-nitrophenol; the base is aninorganic metallic base; the base is sodium hydroxide; and where thesalt is a sodium salt.

Disclosed embodiments include a method for the synthesis of an activatedester of MEF comprising: in a first reaction mixture, mixing anucleophilic alcohol and an acid anhydride in an appropriate solvent;adding a proton scavenger; in a second reaction mixture, mixing MEF anda proton scavenger in an appropriate solvent; and adding the firstmixture to the second mixture. Further embodiments include methodswhere: the alcohol is a phenol with an electron withdrawing substituenton the aryl ring; the alcohol is 4-nitrophenol; the proton scavenger isan organic amine; and wherein the solvent is a polar organic solvent.

Disclosed embodiments include a method for preparing a substitutedaminoalkyl-diketopiperazine including: generating anaminoalkyl-diketopiperazine intermediate; generating an activated esterof MEF; reacting the aminoalkyl-diketopiperazine with the activatedester; and wherein the activated ester of ethylfumarate is reacted insitu without isolation or purification. Further embodiments includemethods: further comprising the step of deprotecting theaminoalkyl-diketopiperazine prior to reaction with the activated ester;wherein the activated ester is a 4-nitrophenyl ester; wherein the stepof generating the activated ester comprises: generating a mixedanhydride of MEF and another acid and reacting the mixed anhydride withan alcohol to produce the activated ester of MEF; and wherein the mixedanhydride is trifluoroacetyl-ethyl-fumarate.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the exemplary embodiments of the inventionwill be had when reference is made to the accompanying drawings, whereinidentical parts are identified with identical reference numerals, andwherein:

FIG. 1 is a scheme showing the synthesis of a substituted3,6-aminoalkyl-2,5-diketopiperazine using an embodiment describedherein.

FIG. 2 shows results from experiments for a series of acetone/watermixtures that were explored to determine the optimum ratio for thereaction embodied in Part A.

FIG. 3 is a graph showing the effects of the p-nitrophenol (p-NP)reactant concentration on quality of compound of the Formula 4 obtained.

FIG. 4 is a graph showing the effects of pH control of Part A [same asabove] reaction versus no pH control during the reaction.

FIG. 5 shows the results from experiments comparing quality of productproduced from Part B with reaction temperature at ambient versuselevated temperatures to 50° C.

FIG. 6 is a graph displaying the results obtained when varying theacetone/water ratios for the TFA deprotection of the diketopiperazineintermediate.

FIG. 7 is a graph depicting data on the characteristics of compound ofthe Formula 4 when the reaction concentration is varied during the TFAdeprotection step.

FIG. 8 is a graph depicting results comparing the charge ofethyl-4-nitrophenylfumarate on quality of compound of the Formula 4obtained by a reaction embodiment disclosed herewith.

FIG. 9 is a graph depicting results obtained using either crude orrecrystallized TFA-DKP when forming the compound of Formula 4.

FIG. 10 is a graph comparing the compound of the Formula 4 overallquality obtained using a conventional method versus employing the insitu methodology described herein.

FIG. 11 is a chemical scheme showing an embodiment of a synthesis for asubstituted aminoalkyl-diketopiperazine.

FIG. 12 is a chemical scheme showing the preparation of an activated MEFmixed anhydride followed by addition to an aminoalkyl-diketopiperazine.

FIG. 13 shows tables of data related to anhydride formation underdifferent conditions: (a) solvent; (b) concentration; (c) equivalents ofTEA; (d) equivalents of CSI; (e) time for CSI addition; and (f) reactiontemperature

FIG. 14 shows tables of data related to substitutedaminoalkyl-diketopiperazine formation under varied conditions: (a) base;(b) solvent; (c) THF/water ratio; (d) reaction time/temperature; (e)effect of addition order.

FIG. 15 shows a chemical scheme for the generation of 4 (R=Et) via anactivated phosphate anhydride of MEF.

FIG. 16 shows tables of data for the scheme shown in FIG. 15 undervariable conditions.

FIG. 17 is a chemical scheme showing the generation of MEF anhydride andsubsequent reaction to give a substituted diketopiperazine.

FIG. 18 shows the results for variable conditions used to generate MEFanhydride.

FIG. 19 shows the results for variable conditions used to react MEFanhydride to give a substituted diketopiperazine.

FIG. 20 is a chemical scheme showing the generation of a MEF mixedanhydride and subsequent reaction to give a substituteddiketopiperazine.

FIG. 21 shows the results for variable conditions used to generate a MEFmixed anhydride.

FIG. 22 shows the results for variable conditions used to react MEFmixed anhydride to give a substituted diketopiperazine.

FIG. 23 shows a chemical scheme for the use of a MEF mixed anhydride forthe generation of a substituted diketopiperazine and subsequentsaponification of the MEF-moiety ester.

FIG. 24 shows results for variable conditions used in the synthesis ofthe saponified substituted diketopiperazine.

FIG. 25 shows a chemical scheme for the use of a MEF mixed anhydride forthe generation of a substituted diketopiperazine after in situdeprotection of the diketopiperazine.

FIG. 26 shows a chemical scheme for the use of a MEF mixed anhydride forthe generation of a substituted diketopiperazine after in situdeprotection of the diketopiperazine and subsequent saponification ofthe ester moiety.

FIG. 27 is a chemical scheme showing the reaction between anaminoalkyl-diketopiperazine and EFC, followed by saponification of theethyl moiety.

FIG. 28 shows the results for 4 acids used to precipitate the productshown in FIG. 27.

FIG. 29 shows results for variable conditions used to couple EFC with anaminoalkyl-diketopiperazine.

FIG. 30 shows a chemical scheme for in situ deprotection of anaminoalkyl-diketopiperazine followed by coupling with EFC.

DETAILED DESCRIPTION

Generally, the aim of optimizing overall yield in a multi-step chemicalsynthesis is accomplished by isolation and purification of eachintermediate molecule prior to subsequent reaction. This approach hopesto avoid loss of the final target due to: a) by-products of the previoussteps reacting with intermediates or starting materials; and b) loss dueto more complicated isolation and purification of the target molecule.

Disclosed embodiments provide methods for the synthesis of substituteddiketopiperazine pharmaceutical excipients via use of in situ generatedintermediates. The embodiments provide results which, counter to generalthought, achieve higher yield and reactor throughput than traditional,isolate-and-purify-type methods. More specifically, embodiments showmethods for the generation and use of fumaroyl intermediates in situ andwithout purification, as well as methods for the generation and use ofaminoalkyl-diketopiperazines in situ and without isolation orpurification. In embodiments disclosed herein, a method is provided forsynthesizing an activated MEF in a simplified one-step process. In anembodiment, an activating group, agent or reactant can be selected froma number of reactants, including, but not limited to diphenylphophorylazide, pivaloyl chloride, chlorosulfonyl isocyanate, p-nitrophenol, MEF,trifluoroacetyl and chlorine, for example, ethyl fumaroyl chloride. Inan exemplary embodiment, ethylfumaroyl chloride is reacted with a phenolcontaining an electron withdrawing moiety (such as p-nitrophenol) toform an activated ester of MEF, the ester is then used in situ as anelectrophile to introduce the fumaryl moiety. In another aspect, anactivated fumarate ester is generated using the sodium salt of areactive alcohol such as 4-nitrophenol. This ester may also be used insitu in coupling reactions.

Turning to the drawings for a better understanding, FIG. 1 shows ascheme for the generation of an ester substitutedaminoalkyl-diketopiperazine. The synthesis of diketopiperazines such asthis usually involves the coupling of the aminoalkyl-diketopiperazinewith the activated ester with after isolation of both penultimateintermediates. Disclosed embodiments illustrate an improved method forthe synthesis of this and similar diketopiperazines resulting inimproved yields and reactor throughput.

EXAMPLES

Coupling of ethyl fumaroyl chloride and 4-nitrophenol: A 1 L 4-neckround bottomed flask was charged with 11.20 g (80.51 mmol) of4-nitrophenol, 90 mL of water, and 69 mL of acetone. While stirringunder nitrogen, a solution of 12.80 g (120.8 mmol) of sodium carbonatein 90 mL of de-ionized water was added to the reaction. Ethyl fumarylchloride (EFC) (17.0 mL, d=1.16 g/mL, 121 mmol) in 21 mL of acetone wasadded to the mixture using an addition funnel. An exotherm of 25-33° C.was observed during the EFC addition. As EFC addition progressed, thereaction mixture faded from yellow to colorless. At the end of the EFCaddition, the reaction pH was 7-7.5. Approximately 15 minutes after theEFC addition was complete, the reaction was diluted with 450 mL ofde-ionized water. A precipitate formed at 27° C. The mixture was heldfor 15 minutes, and then the solids were isolated, washed withde-ionized water (3×220 mL), and dried in a 50° C. vacuum oven for 1hour. The product was analyzed for weight percent purity.

The coupling of EFC and 4-nitrophenol to generateethyl-4-nitrophenylfumarate was evaluated in a total of eightexperiments. The base and solvent system were held constant acrossexperiments; the reaction and quench times were varied. When no time isprovided for the reaction between EFC and 4-nitrophenol, the productyield appears to increase. However, this may be due to isolating excesssodium carbonate along with the product; the low wt % purities of thesematerials support this hypothesis. Reaction times of 15-60 minutes gavegood ethyl-4-nitrophenylfumarate yield and purity (>96% and >94 wt %,respectively). Similarly, quench times of 15-45 minutes gave goodproduct quality.

Scale (mmol) Time Quench Time Yield (%) Weight 14 45 30 97 96.22 14 6045 96 96.28 36 15 15 99 98.55 36 15 15 97 90.08 36 0 15 101 87.5 36 3015 122 87.94 36 0 30 112 82.85 81 15 15 96 94.94

Coupling of Ethyl Fumaryl Chloride and 4-Nitrophenol Followed by in situuse with Deprotected DKP

Part A (in situ ethyl-4-nitrophenylfumarate formation): A 1 L, 3-neckround bottom flask was equipped with a magnetic stirrer, temperaturereadout/controller, and an addition funnel with a nitrogen inlet. Theexhaust gas was vented to a caustic scrubber. p-nitrophenol (9.18 g,0.066 mol) and acetone (10 mL) were charged to the flask. Sodiumhydroxide (2.90 g, 0.073 mol) dissolved in water (25 mL) was then addedto the reaction mixture. During the sodium hydroxide addition, anexotherm of −^(|)15° C. was observed and the reaction mixture changedfrom a clear yellow solution to yellowish orange suspension/slurry.After the addition was complete, the reaction mixture was cooled to 20°C. and EFC (8.78 g, 0.054 mol) in acetone (10 mL) was added via additionfunnel over 5-10 minutes. During the EFC addition, an exotherm of−^(|)15° C. was observed and the reaction mixture changed from orange toyellow; a solid was observed about 20 minutes after addition. The pH ofthe reaction mixture at the end of the EFC addition was 7-8. Thereaction mixture was stirred at room temperature for an hour beforeadditional acetone (30 mL) was added to dissolve the precipitated 022.

Part B (crude 4 formation): In a 250 mL Erlenmeyer flask, a solution ofsodium hydroxide (8.82 g, 0.44 mol) in water (25 mL) was diluted withacetone (10 mL). Aminoalkyldiketopiperazine (Formula 1: R₁═R₂═H; n=3)(7.98 g, 0.021 mol) was charged to the Erlenmeyer flask. The neutralizeddiketopiperazine solution was charged to the round bottom flaskcontaining the in situ ethyl-4-nitrophenylfumarate; the diketopiperazineflask was rinsed into the reactor with water (5 mL). The reactionmixture was heated to 50° C., held at temperature for one hour, cooledto ˜30° C., and then quenched with water (50 mL). The resulting solidswere collected by filtration, washed with water (2×100 mL) and acetone(2×100 mL) and dried in a vacuum oven at 50° C. overnight. The solidswere analyzed using HPLC TM5466. Reaction yield, wt % purity, and area %purity were monitored.

FIG. 2 shows the results for a series of acetone/water mixtures thatwere explored to determine the optimum ratio for the reaction describedin Part A (reaction between ethyl fumaroyl chloride and p-nitrophenol).The reaction does not proceed well in the absence of water. The resultssuggest that an acetone/water ratio of 1:1.25 provides a balance of highyield and purity. This is surprising as sodium hydroxide in water is acommon method of saponification of esters but the ester forms andremains for future reaction.

FIG. 3 shows a graph of the results of reaction concentration on qualityof 4 obtained. The intermediate conditions tested (2.8 mL solvent/mmolp-NP) gave a good balance of substituted aminoalkyl-diketopiperazineyield, purity, and reactor throughput. At high concentration, reactorthroughput increased, but substituted aminoalkyl-diketopiperazines yieldand purity suffered; at low concentration, wt % purity was slightlylower. The intermediate concentration tested yielded about 36 g of 4(R=Et) was per 1 L of reactor space, about 30% better throughput thanthe conventional reaction.

FIG. 4 shows a graph of the results of controlling the pH of Part Areaction versus no pH control during the reaction. No significantdifference in 4 yield or purity was obtained when reaction pH wascontrolled at 7.5 during EFC addition versus experiments where pH wasnot controlled.

FIG. 5 shows the results of comparing quality of product produced fromPart B with reaction temperature at ambient versus elevated to 50° C.The results indicate that elevated temperatures provide better quality.

These studies demonstrated that EFC and p-NP can be combined to form anactivated ester, then treated with aminoalkyl-diketopiperazines usingsodium hydroxide as a base, to form crude substitutedaminoalkyl-diketopiperazines in yields and purities comparable to knownprocesses (isolating the penultimate intermediates and utilizing Na₂CO₃for the final coupling), and with better reactor throughput. Resultswith sodium hydroxide were comparable to those obtained using sodiumcarbonate.

In situ TFA-DKP deprotection followed by in situ use ofethyl-4-nitrophenyl fumarate: Part A (in situethyl-4-nitrophenylfumarate formation): A 1 L, 3-neck round bottom flaskwas equipped with a magnetic stirrer, temperature readout/controller,and an addition funnel with a nitrogen inlet. The exhaust gas was ventedto a caustic scrubber. p-Nitrophenol (9.18 g, 0.066 mol) and acetone (10mL) were charged to the flask. Sodium hydroxide (2.90 g, 0.073 mol)dissolved in water (25 mL) was then added to the reaction mixture.During the sodium hydroxide addition, an exotherm of −^(|)15° C. wasobserved and the reaction mixture changed from a clear yellow solutionto a yellowish orange suspension/slurry. After the addition wascomplete, the reaction mixture was cooled to 20° C. and EFC (8.78 g,0.054 mol) in acetone (10 mL) was added via addition funnel over 5-10minutes. During the EFC addition, an exotherm of −^(|)15° C. wasobserved and the reaction mixture changed from yellowish orange to ayellow suspension/slurry. The reaction mixture pH at the end of the EFCaddition was 7-8. The reaction mixture was stirred at room temperaturefor an hour before additional acetone (15 mL) was added to dissolve theprecipitated ethyl-4-nitrophenylfumarate.

Part B (crude 4 formation): A 250 mL round bottom flask was charged withthe protected diketopiperazine (Formula 1, R₁═R₂═TFA; n=3, TFA-DKP)(9.68 g, 0.022 mol) and acetone (25 mL). Sodium hydroxide (2.16 g, 0.054mol) dissolved in water (30 mL) was added to the TFA-DKPslurry/suspension. The mixture was stirred for 30 minutes at roomtemperature. The resulting clear, yellow solution was added to the flaskcontaining the in situ ethyl-4-nitrophenylfumarate. The TFA-DKP flaskwas rinsed into the reactor with water (10 mL). The reaction mixture washeated to 45° C., held at temperature for one hour, cooled to −^(|)30°C., and quenched with water (50 mL). The resulting solids were collectedby filtration, washed with water (2×100 mL) and acetone (2×100 mL) anddried in a vacuum oven at 50° C. overnight. The solids were analyzedusing HPLC TM5466. Reaction yield, wt % purity, and area % purity weremonitored.

FIG. 6 shows a graph displaying the results obtained when varying theacetone/water ratios for the TFA deprotection of the diketopiperazineintermediate. The results suggested that an acetone/water ratio of1:1.12 for the TFA-DKP resulted in the highest 4 (R=Et) yield andpurity.

FIG. 7 shows a graph depicting the results of 4 quality when reactionconcentration is varied during the TFA deprotection step. One of theintermediate conditions tested (7.95 mL solvent/mmol TFA-DKP) gave thebest balance of 4 (R=Et) yield, purity, and reactor throughput. Athigher concentration, reactor throughput was increased, but 4 (R=Et) wt% purity suffered; lower concentrations gave comparable yield and puritybut poorer reactor throughput. A reaction concentration of 7.95 mLsolvent/mol TFA-DKP gives ˜40 g of 4 per 1 L of reactor space, about 40%better throughput than the current 4 (R=Et) reaction.

FIG. 8 is a graph of the results comparing the charge ofethyl-4-nitrophenylfumarate on quality of 4 obtained. The resultsindicate that there is no significant increase in overall quality whenincreasing the charge above 2.5 molar equivalents.

FIG. 9 is a graph of the results obtained using either crude orrecrystallized TFA-DKP when forming 4. The results indicate thatintermediate TFA-DKP purity had a negligible effect on 4 quality.

These studies demonstrated that in situ ethyl-4-nitrophenylfumarate canbe coupled with deprotected TFA-DKP using sodium hydroxide as the base.Compared to the current process, the best conditions identified in thisstudy give 4 in comparable purity but with better yield and reactorthroughput (40% more).

FIG. 10 is a graph comparing 4 overall quality obtained using aconventional method versus employing the in situ methodology. From thisgraph it is clear that the in situ scheme generates a higher qualityproduct and significantly increases reactor throughput.

The following table shows the results from couplingethyl-4-nitrophenylfumarate with an aminoalkyl-diketopiperazine. Bottomsix reactions were carried out using in situethyl-4-nitrophenylfumarate, using EFC p-NP and TFA-DKP.

Mass Moles of % Yield from 1 L diketopiperazine Base (corrected) flask.053 Na₂CO₃ 88.9 28 .085 Na₂CO₃ 84 36.37 .022 NaOH 90 35.6 .022 NaOH 9337 .028 NaOH 91 47 .022 NaOH 83 35.8 .028 NaOH 82 46 .028 NaOH 90 47.028 NaOH 73 42 .028 NaOH 90 47 .028 NaOH 94 53.84 .028 NaOH 99 56.68.022 NaOH 100 44 .022 NaOH 94 41.52 .022 NaOH 92 40.32 .028 NaOH 11364.67

Example 4

Experimental Preparation of 4 from MEF, TFAA and p-NP, NaOH for couplingFIG. 11: Part A: A 250 mL 3-neck round bottom flask was equipped with amagnetic stirrer, a temperature readout/controller, and an additionfunnel with a nitrogen head. The exhaust gas was vented to a causticscrubber. The flask was charged with p-nitrophenol (p-NP, 10 g) andtrifluoroacetic anhydride (TFAA, 16.61 g, 11 mL) and stirring wasinitiated. The resulting yellow slurry was treated with triethylamine(TEA, 600 μL). An exotherm of −^(|)12° C. was observed after the TEAaddition. The solution was stirred for about 30 minutes (until clear, anindication that formation was complete).

Part B (in situ ethyl-4-nitrophenylfumarate formation): A 250 mL 4-neckround bottom flask was equipped with a magnetic stirrer, a temperaturereadout/controller, an addition funnel with a nitrogen head and a refluxcondenser. Monoethyl fumarate (MEF, 10.36 g) and acetone (9 mL) werecharged to the flask. TEA (16.33 mL) was charged to the flask; anexotherm of −^(|)15° C. was observed after the TEA addition. Theresulting clear solution was cooled to 20° C. and the Part A solutionwas slowly added via addition funnel. The reaction temperature wasmaintained below 30° C. for the duration of the addition. The Part Aflask was rinsed with acetone (3 mL), and the rinse added to thereaction flask. The reaction mixture was stirred for 30 minutes whilemaintaining the temperature between 20-30° C.

Part C (crude 4 (R=Et) formation): A 250 mL round bottom flask wascharged with TFA-DKP (12.66 g) and acetone (25 mL). Sodium hydroxide(2.83 g) dissolved in water (30 mL) was added to the TFA-DKP slurry. Themixture was stirred at room temperature for about 30 minutes. Theresulting clear, yellow solution was added to the flask containing thein situ ethyl-4-nitrophenylfumarate. The TFA-DKP flask was rinsed intothe reactor with water (10 mL), and additional acetone (23 mL) and water(35 mL) were charged to the reaction mixture. The reaction mixture washeated to 45° C., held at temperature for one hour, cooled to −^(|)30°C., quenched with water (50 mL) and stirred for additional 30 minutes.The resulting solids were collected by filtration, washed with water(2×100 mL) and acetone (2×100 mL) and dried in a vacuum oven at 50° C.overnight. The solids were analyzed using HPLC TM5466. Reaction yield,wt % purity, and area % purity were monitored.

In situ ethyl-4-nitrophenylfumarate was first generated from MEF andp-nitrophenyl trifluoroacetate, then coupled with deprotected TFA-DKP(2). The resulting crude 4 (R=Et) was obtained in 63% yield and 85 wt %purity. The initial conditions tested gave −^(|)36 g of 4 per 1 L ofreactor space, about 35% better throughput than the current process.

Substituting THF for acetone gave lower product yield, but comparablepurity; the trans isomer content was elevated in this sample. Use ofadditional TFAA in the in situ ethyl-4-nitrophenylfumarate formationstep failed to improve 4 (R=Et) yield or purity.

g per TFAA % 1 L % % % Sample ID Equivalents Solvent Yield flask TransWt Area D733-47A 1.1 Acetone 63 36.4 52.97 84.73 91.79 D733-47T 1.1 THF38 21.56 71.45 84.62 88.15 D733-67 1.25 Acetone 23 13.22 59.31 56.1751.25

From this table it is clear that the in situ generation and use of theactivated MEF gave good yield and, more importantly, improvedthroughput.

Example 5

A 500 mL, 3-neck round bottom flask was equipped with a magneticstirrer, temperature readout/controller, and an addition funnel with anitrogen inlet. The exhaust gas was vented to a caustic scrubber.Monoethyl fumarate (MEF, 5 g), dry dichloromethane or THF (10 mL), andtriethylamine (TEA, 12 mL) were charged to the flask. An exotherm wasobserved during the TEA addition. The clear reaction mixture was cooledto 5° C. in an ice bath. A solution of chlorosulfonyl isocyanate (CSI,4.96 g) in 10 mL of dry dichloromethane was added over 20-30 minutes.After the addition was complete, the reaction mixture was held below 10°C. for 3 hours. For reactions using DCM, the crude MEF anhydride wasisolated by removing the solvent in vacuo. For reactions using THF asthe solvent, the MEF anhydride was used without further manipulation.

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with 2 (5.47 g) and a solution of sodium carbonate (8.90 g) inwater (80 mL). The activated anhydride obtained in step 1 was dissolvedin THF (80 mL), and added to the flask. The reaction mixture was stirredat room temperature overnight. The resulting solids were collected byfiltration, washed with water (50 mL) and acetone (20 mL) and dried in avacuum oven at 50° C. overnight. The solids were analyzed using HPLCTM5466. Reaction yield, wt % purity, and area % purity were monitored.Assay-corrected yield was calculated by multiplying the yield by the wt% purity.

FIG. 12 shows a chemical scheme for the generation of an activated MEFanhydride and subsequent reaction with a diketopiperazine.

FIG. 13a-f shows the results for MEF anhydride formation under variedconditions. Solvent, reaction concentration, amounts of TEA and CSI,addition time, and addition temperature were explored. FIG. 13a suggeststhat THF gives MEF activated anhydride in higher wt % purity than DCMand comparable assay-corrected yield. The other tested parameters (FIGS.13b-f ) did not affect assay-corrected yield or area % purity.

FIG. 14a-e shows the results for using the CSI-MEF anhydride and anaminoalkyl-diketopiperazine. The MEF activated anhydride was formed insitu using THF as the solvent, and then added to a basic solution of 2to generate 4 (R=Et).

Three bases were evaluated: triethylamine, sodium carbonate and sodiumhydroxide and the results shown in FIG. 14a . Sodium hydroxide produced4 (R=Et) in higher assay-corrected yield than sodium carbonate;triethylamine was not suitable for this reaction because no material wasobtained after a 72 hour reaction time.

Three solvents were evaluated: THF, DCM and acetone and the resultsshown in FIG. 14b . THF gave 4 (R=Et) in higher assay-corrected yieldand purity than the other tested solvents. In addition, severaldifferent water/THF mixtures were explored because the reaction did notproceed well in the absence of water. However, the addition of water didnot appear to improve yield or product quality (FIG. 14c ).

Different reaction time and temperature combinations were also explored.High reaction temperature gave little 4; at room temperature,assay-corrected yields were increased with increasing time up to 18 hrs(FIG. 14d ).

FIG. 15 shows a chemical scheme for the generation of an activated MEFanhydride and subsequent reaction with a diketopiperazine.

Example: 4 (R=Et) Preparation using an Activated MEF Phosphate Anhydride

A 500 mL, 3-necked round bottom flask was equipped with a magneticstirrer, temperature readout/controller, and addition funnel with anitrogen head. The exhaust gas was vented to a caustic scrubber.Mono-ethyl fumarate (MEF, 5 g), THF (15 mL), and triethylamine (TEA, 10mL) were charged to the flask. An exotherm was observed during the TEAaddition. Diphenylphosphoryl azide (DPPA, 9 mL) was added to thereaction mixture, followed immediately by the addition of 2 solution(28.21 g) dissolved in a solution of sodium carbonate (18.23 g) andwater (60 mL). The flask containing the 016 was rinsed into the reactionmixture with water (10 mL). The reaction mixture was stirred at roomtemperature overnight. The resulting solids were collected byfiltration, washed with water (2×100 mL) and acetone (2×50 mL) and driedin a vacuum oven at 50° C. overnight. The solids were analyzed usingHPLC TM5466.

Parameter Screen for 4 (R=Et) Syntheses via Activated MEF PhosphateAnhydride

FIG. 16a shows that THF gave 4 (R=Et) in higher wt % purity andassay-corrected yield than the other tested solvents. THF was used asthe solvent for further studies. The effect of base (sodium carbonatevs. sodium hydroxide) was also evaluated. FIG. 16b shows that sodiumhydroxide produced 4 (R=Et) in higher wt % purity (80%); however, sodiumcarbonate gave higher assay corrected yield. Both wt % purity andassay-corrected yields were increased with increasing time, regardlessof the base used FIGS. 16c and d. FIG. 16e shows that the use of 2 insolid form significantly increased 4 (R=Et) wt % purity compared to 4(R=Et) made from an acetic acid solution of 2.

FIG. 17 shows a chemical scheme for the generation of a dimeric MEFanhydride and subsequent reaction with an aminoalkyl-diketopiperazine.

Example: Dimeric MEF Anhydride Preparation

A 500 mL, 3-neck round bottom flask was equipped with a magneticstirrer, temperature readout/controller, and an addition funnel with anitrogen inlet. The exhaust gas was vented to a caustic scrubber.Monoethyl fumarate (MEF, 20 g), dry dichloromethane (DCM, 25 mL), anddry triethylamine (TEA, 20 mL) were charged to the flask. An exothermwas observed during TEA addition. The clear reaction mixture was cooledto −25° C. in a dry ice/acetone bath. A solution of chlorosulfonylisocyanate (CSI, 9.8 g, 6.1 mL) in 10 mL of dry dichloromethane wasadded over 15-20 minutes. The temperature of the reaction mixture wasmaintained below 0° C. during addition. After the addition was complete,the reaction mixture was held below 10° C. for 6 hours. Water (200 mL)was added to the reaction flask. The layers were separated, and theaqueous phase was extracted with dichloromethane (2×200 mL). The organicphases were combined, dried over sodium sulfate, filtered andconcentrated in vacuo. The resulting dimeric MEF anhydride was obtainedin 94% yield and was used without further purification.

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with solid 2 (5.47 g) and a solution of sodium carbonate (8.80g) in water (60 mL). THF (20 mL) was also added and the mixture wasstirred until a clear solution was obtained. Dimeric MEF anhydride (8.97g) was dissolved in THF (32 mL), and added to the reaction flask byaddition funnel over 10-15 minutes. The reaction mixture was stirred atroom temperature for 6 h.

The reaction mixture was quenched with water (50 mL) and stirring wascontinued for an additional 45 minutes. The resulting solids werecollected by filtration, washed with water (2×50 mL) and acetone (50mL), and dried in a vacuum oven at 50° C. overnight. The solids wereanalyzed using HPLC TM5466.

FIG. 18a-d shows the results for the synthesis of dimeric MEF anhydridewhen several conditions are varied. Solvent (a), CSI additiontemperature (b), hold temperature after CSI addition (c), and reactiontime (d) were explored. DCM appeared to provide superior resultscompared to THF.

FIG. 18b shows the effect of CSI addition temperature. CSI addition wasstarted at 5° C. and the reaction temperature was maintained below 20°C. throughout addition and CSI addition was started at −25° C. and thereaction temperature was maintained below 0° C. throughout addition. Thetemperature conditions for dimeric MEF anhydride preparation did notaffect 4 yield and purity.

The reaction hold temperature after CSI addition was also explored (FIG.18c ). The results suggest that lower hold temperatures gave 4 in betteryield with comparable purity. Different reaction times were exploredFIG. 18d . The intermediate conditions tested (6 hours stirring) gave agood balance of 4 yield and purity. Short reaction times (i.e., 3 hours)were not sufficient for the reaction to complete, but extended times(i.e., 17 hours) permitted product degradation.

Taken together, the results suggested that the best conditions fordimeric MEF anhydride preparation included using DCM as the solvent,maintaining low temperatures during CSI addition, and holding for 6 hafter CSI addition. Therefore, these conditions were used for furtherevaluation.

Parameters Screened for 4 Formation

Dimeric MEF anhydride was formed using the above conditions, and thenconverted to 4. The effects of various coupling conditions wereevaluated and results shown in FIG. 19a -f. The condition variablesinclude base choice, (a) solvent, (b) solvent-water ratio, reactiontemperature and reaction time (c) and (d); Na₂CO₃ charge (e) and use ofsolid or liquid form for the amine (f).

FIG. 19a shows the results where five solvents were evaluated: THF,acetone, DCM, ethyl acetate (EtOAc) and acetonitrile (ACN). THF gave 4in higher yield and purity than the other tested solvents. In addition,several different water/THF mixtures were explored (FIG. 19b ) becausethe reaction did not proceed well in the absence of water. The resultssuggested that 4 (R=Et) purity was maximized when a 1:1 THF/water ratiowas used. Different reaction time and temperature combinations were alsoexplored using two bases (sodium carbonate and sodium hydroxide). Areaction time of 3-6 hours at room temperature using Na₂CO₃ gave 4(R=Et) in 90% yield with 88 wt % purity (FIG. 19c ). Neither reactiontime nor temperature affected 4 (R=Et) yield and purity when NaOH wasused as the base, but yields were lower compared to Na₂CO₃ (FIG. 19d ).In short, the highest 4 (R=Et) yield obtained during this study was ˜91%with 90 wt % purity using solid 2. Using 2 as an aqueous acetic acidsolution gave 2 in good yield (90%), but with low purity (51%).

Example: MEF Mixed Anhydride Preparation

A 1 L, 4-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogeninlet. The exhaust gas was vented to a caustic scrubber. Monoethylfumarate (MEF, 30 g), dichloromethane (DCM) or tetrahydrofuran (THF)(200 mL), and triethylamine (TEA, 45 mL) were charged to the flask. Anexotherm was observed during the TEA addition. The clear reactionmixture was cooled to −25° C. in dry ice/acetone bath. A solution ofpivaloyl chloride (39.2 g, 40 mL) in 20 mL of DCM or THF was added over15-20 minutes. The reaction temperature was maintained below −10° C.during addition. After the addition was complete, the reaction mixturewas slowly brought to room temperature and was stirred for two hours.The resulting solids were removed by filtration though celite. Thefilter cake was washed with DCM or THF (2×100 mL) and acetone (50 mL).The filtrate was concentrated in vacuo to give the MEF mixed anhydridein 96% yield. This material was used without further purification.

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with solid 2 (5 g) and a solution of sodium carbonate (8.72 g)in water (60 mL). THF (20 mL) was also added and solution was stirreduntil clear. The MEF mixed anhydride (8 g) was dissolved in THF (25 mL),and added to the reaction flask via addition funnel over 5-10 minutes.The reaction mixture was stirred at room temperature for 3 h, thenquenched with water (100 mL) and stirred for an additional 30 minutes.The resulting solids were collected by filtration, washed with water(2×80 mL) and acetone (2×80 mL), and dried in a vacuum oven at 50° C.overnight. The solids were analyzed using HPLC TM5466. Reaction yield,wt % purity, and area % purity were monitored.

FIG. 20 shows a chemical scheme for the generation of a MEF mixedanhydride and subsequent reaction to give a substituteddiketopiperazine.

FIG. 21a-d shows results for variable conditions used to generate a MEFmixed anhydride generally according to FIG. 20 and the effect on 4(R=Et) production. (a) solvent, (b) pivaloyl chloride additiontemperature, (c) hold temperature after pivaloyl chloride addition, and(d) reaction time were explored. Different combinations of pivaloylchloride addition temperature, hold temperature and time were explored.The results suggest that all the conditions tested gave 4 (R=Et) incomparable yield and purity.

Parameters Screened for 4 (R=Et) Formation

MEF mixed anhydride was formed using the conditions described above, andthen converted to 4 (R=Et). FIG. 22a-f shows the results where variouscoupling conditions were evaluated, including base choice, solvent,reaction temperature and reaction time. Three solvents were evaluated:THF, acetone, and acetonitrile (ACN). THF gave 4 (R=Et) in better puritythan the other tested solvents (FIG. 22a ). Different reaction time andtemperature combinations were also explored using two bases (sodiumcarbonate and sodium hydroxide). A reaction time of 3 hours at roomtemperature using Na₂CO₃ gave 4 (R=Et) in 93% yield with 89 wt % purity(FIG. 22b ). Lower yields and purities were obtained when NaOH was usedas base (FIGS. 22c and d ). The use of an aqueous acetic acid solution(2 L) significantly decreased 4 (R=Et) yield and purity compared to useof 2 as a solid (FIG. 22e ). Using NaOH instead of Na₂CO₃ as the 2 Lbase resulted in even lower 4 (R=Et) yields; however, unlike the resultobserved with 2S, increasing the 2 L sodium hydroxide charge increased 4(R=Et) yield and purity (FIG. 22f vs. 22 d).

FIG. 23 shows a chemical scheme for the use of a MEF mixed anhydride forthe generation of a substituted diketopiperazine and subsequentsaponification of the MEF-moiety ester.

Preparation of 4 (R=Et) using Na₂CO₃ as Base

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with solid 2 (5 g) and a solution of sodium carbonate (8.99 g)in water (60 mL). THF (20 mL) was also added and the solution wasstirred until clear. MEF mixed anhydride (8 g) was dissolved in THF (25mL), and added to the reaction flask via addition funnel over 5-10minutes. The reaction mixture was stirred at room temperature for 3 h tofacilitate in situ formation of 4 (R=Et). The reaction mixture wasquenched with water (100 mL) and stirring was continued for anadditional 30 minutes. Methanol (50 mL) was added to the reactionmixture and the reaction mixture was heated to reflux. Sodium hydroxide(5.45 g) solution in water (50 mL) was added to the reaction mixture viaaddition funnel over 5 minutes. The mixture was heated for about 10minutes (until clear, an indication that 4 (R=Et) saponification wascomplete giving 4 (R═H)), and then cooled to 25° C. Concentrated HCl (35mL) was added and reaction mixture was stirred for 2 hours at roomtemperature. The resulting solids were collected by filtration, washedwith water (2×80 mL) and acetone (2×80 mL), and dried in a vacuum ovenat 50° C. overnight. The solids were analyzed using HPLC TM5478.Reaction yield, wt % purity, and area % purity were monitored.

Preparation of 4 using NaOH as Base

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with solid 2 (5 g) and a solution of sodium hydroxide (0.65 g)in water (60 mL). THF (20 mL) was also added and the mixture was stirreduntil clear. MEF mixed anhydride1 (8 g) was dissolved in THF (25 mL),and added to the reaction flask via addition funnel over 5-10 minutes.The reaction mixture was stirred at room temperature for 30 minutes tofacilitate in situ 4 formation, then quenched with water (100 mL) andstirred for an additional 30 minutes.

Methanol (50 mL) was added to the reaction mixture and the reactionmixture was heated to reflux. A solution of sodium hydroxide (4.75 g) inwater (50 mL) was added to the reaction mixture via addition funnel over5 minutes. The mixture was heated for approximately 10 minutes (untilclear, an indication that saponification was complete giving 4 (R═H)),and then cooled to 25° C. Concentrated HCl (20 mL) was added and thereaction mixture was stirred for 2 hours at room temperature. Theresulting solids were collected by filtration, washed with water (2×80mL) and acetone (2×80 mL), and dried in a vacuum oven at 50° C.overnight. The solids were analyzed using HPLC TM5478. Reaction yield,wt % purity, and area % purity were monitored.

Parameters Screened for 4 (R═H) Formation

MEF mixed anhydride was prepared, 1 converted to in situ 4 (R=Et), andthen to 4 (R═H) in a single reaction vessel. FIG. 24a-g shows resultsfor the effects of various 4 (R=Et) coupling conditions, including basechoice, reaction temperature and reaction time. Different reaction timeand temperature combinations were explored using two bases (sodiumcarbonate and sodium hydroxide). A coupling reaction time of 3 hours atroom temperature using Na₂CO₃ followed by saponification with sodiumhydroxide gave 4 (R═H) in 89% yield and 78 wt % purity; increasingtemperature and decreasing time decreased 4 (R═H) yield and purity (FIG.24a ). When NaOH was used as the coupling base, lower 4 (R═H) yield andpurity were obtained with increasing reaction time at a fixed reactiontemperature, and lower yield and purity were obtained with increasingreaction temperature at a fixed reaction time (FIG. 24b ). Yield andpurity were unaffected by NaOH charge (FIG. 24c ). A slight decrease inyield was observed when the reaction was not quenched with water (FIG.24d ). Eliminating methanol from the saponification reaction did notaffect 4 (R═H) yield or purity (FIG. 24e ); however, reaction filtrationsuffered in the absence of methanol. The use of an 016 aqueous aceticacid solution (2 L) decreased 4 (R═H) yield and purity compared to useof 2 solid (FIG. 24f ). Using NaOH instead of Na₂CO₃ as the 016 L baseresulted in even lower 4 (R═H) yields; however, unlike the resultobserved with 2S, increasing the 2 L sodium hydroxide charge increased 4(R═H) yield and purity (FIG. 24g vs. FIG. 24c ).

FIG. 25 shows a chemical scheme for the use of a MEF mixed anhydride forthe generation of a substituted diketopiperazine after in situdeprotection of the diketopiperazine.

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,a temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with TFA-DKP (5 g), THF (30 mL) and water (30 mL) and stirringwas initiated. A solution of sodium hydroxide (1.20 g) in water (30 mL)was added and the solution was stirred for about 15 minutes (untilclear, an indication that TFA-DKP deprotection was complete). The MEFmixed anhydride1 (7.53 g) was dissolved in THF (30 mL) and added to thereaction flask via addition funnel over 5-10 minutes. The reactionmixture was stirred at room temperature for 30 minutes, then quenchedwith water (50 mL) and stirred for an additional 15 minutes. Acetone (15mL) was added and the reaction mixture was stirred for additional 15minutes. The resulting solids were collected by filtration, washed withwater (2×70 mL) and acetone (3×70 mL), and dried in a vacuum oven at 50°C. overnight. The solids were analyzed using HPLC TM5466. Reactionyield, wt % purity, and area % purity were monitored. Assay-correctedyield was calculated by multiplying the yield by the wt % purity.

Sample ID % Yield % Trans % Area % Wt D698-45 84 18.81 95.57 86.28D698-47 86 27.74 95.28 85.15

TABLE 2 Analysis results of 004 obtained from coupling of TFA-DKP/mixedanhydride Reac- g per % Assay tion % 1 L % % corrected Sample ID Conc.*Solvent Yield flask Trans Wt yield D698-45 30 THF 84 19.08 18.81 86.2872.5 D698-47 30 THF 86 19.54 27.74 85.15 73.2 D733-27T 6.87 THF 99 39.4866.07 68.47 67.8 D733-23T 5.86 THF 75 38.38 66.33 69.19 51.9 D733-27A6.87 Acetone 88 35.04 54.58 75.06 66.1 D733-23A 5.86 Acetone 84 43.5655.45 62.03 52.1

An MEF mixed anhydride was prepared and then coupled with deprotectedTFA-DKP. The resulting crude 4 (R=Et) was obtained in 85% yield and 86wt % purity. The trans isomer content was low; this was because theTFA-DKP starting material contained only the cis isomer. Solventscreening studies suggested that THF and Acetone gave comparable assaycorrected yields. The trans isomer content increased with increasingreaction concentration and was highest when THF was used as the solvent.

FIG. 26 shows a chemical scheme for the use of a MEF mixed anhydride forthe generation of a substituted diketopiperazine after in situdeprotection of the diketopiperazine and subsequent saponification ofthe ester moiety.

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with TFA-DKP (5 g), THF (30 mL) and water (30 mL) and stirringwas initiated. A solution of sodium hydroxide (1.20 g) in water (30 mL)was added and the solution was stirred for about 15 minutes (untilclear, an indication that TFA-DKP deprotection was complete). MEF mixedanhydride1 (7.53 g) was dissolved in THF (30 mL), and added to thereaction flask via addition funnel over 5-10 minutes. The reactionmixture was stirred at room temperature for 30 minutes to facilitatefumaramide bond formation, then quenched with water (50 mL) and stirredfor an additional 15 minutes. Methanol (50 mL) was added and thereaction mixture was stirred for additional 15 minutes. The reactionmixture was heated to reflux (−^(|)69° C.). Sodium hydroxide (4.00 g) inwater (50 mL) was added to the reaction mixture via addition funnel over5 minutes. The mixture was heated for about 10 minutes (until clear, anindication that saponification was complete), cooled to 25° C.

Concentrated HCl (30 mL) was added and reaction mixture was stirred for2 hours at room temperature. The resulting solids were collected byfiltration, washed with water (2×80 mL) and acetone (2×80 mL), and driedin a vacuum oven at 50° C. overnight. The solids were analyzed usingHPLC TM5478. Reaction yield, wt % purity, and area % purity weremonitored.

MEF mixed anhydride1 was synthesized, and then coupled with deprotectedTFA-DKP, saponified, and precipitated in a single reaction vessel. Theresulting crude 4 (R═H) was obtained in −^(|)85% yield and −^(|)75 wt %purity. The solvent used to make the mixed anhydride had no influence oncrude 4 (R═H) quality.

Sample Solvent for Name anhydride % Yield % Trans % Area % Wt 1 THF 8753.9 87.67 75.54 2 DCM 85 53.7 87.88 75.59 3 THF 85 54.1 87.75 75.33 4DCM 83 53.9 85.93 72.64

The following table shows data for recrystallized 4 (R═H) obtained fromcoupling TFA-DKP with MEF mixed anhydride. Specifications are shown inblue. Out of specification results are shown in red.

% trans % % 354 396 450A 466A 466B 484 788 806 Sample ID (53-63) YieldWt ≧92 0.3 0.45 0.3 0.45 0.15 0.75 0.45 0.20 5 75.8 84 92.0 0.09 0.010.06 0.02 0.85 0.10 0.32 0.01 6 61.5 79 92.6 0.09 0.03 0.04 0.01 0.020.09 0.56 0.01 7 62.2 70 91.7 0.11 0.03 0.04 0.01 0.98 0.17 0.58 0.05 861.5 69 92.0 0.07 0.01 0.04 0 0.72 0.09 0.55 0

The coupling of MEF mixed anhydride and TFA-DKP, followed bysaponification and precipitation to crude 4 (R═H) in a single vessel,was evaluated. The resulting crude 4 (R═H) was obtained in good yieldand purity. Crude 4 (R═H) recrystallization gave material in good purity(NLT 92 wt %).

The following tables show the results for coupling ethyl fumaroylchloride and an aminoalkyl-diketopiperazine under varying conditions.

Hold Serial Water Time Wash Water, Area Na₂CO₃ (7.0 Acetone AdditionAfter Acetone Percent Notebook Formula mL/g Eq. Acid (1 mL/1 mL TempAddition (mL/g Yield Purity Number Multiplier Na₂CO₃ Chloride water) (°C.) (min) 004 theo.) (%) (%) 470-123 1.05 75 6.0 75 25 30 0 86.8 70.47470-125 1.00 71 3.0 71 25 180 10 54.8 63.97 470-127 1.05 75 6.0 75 25180 10 70.3 70.78 470-132 1.00 71 6.0 71 25 30 10 64.3 76.28 470-1341.05 75 3.0 75 10 30 0 76.8 66.45 470-138 1.00 71 3.0 71 10 30 10 61.861.59 470-140 1.05 75 6.0 75 10 30 10 56.9 71.59 470-142 1.00 71 6.0 7110 30 0 84.8 67.40

Hold Serial Water Time Wash Water, Area Na₂CO₃ (7.0 Acetone AdditionAfter Acetone Percent Notebook Formula mL/g Eq. Acid (1 mL/1 mL TempAddition (mL/g Yield Purity Number Multiplier Na₂CO₃ Chloride water) (°C.) (min) 004 theo.) (%) (%) 470-147 1.05 75 3.0 75 25 180 0 92.8 62.96470-163 1.00 71 6.0 71 10 180 10 57.4 73.68 470-150 1.00 71 3.0 71 10180 0 82.4 60.19 479-159 1.05 75 3.0 75 25 30 10 67.9 66.30 470-161 1.0071 3.0 71 25 30 0 85.9 65.69 470-172 1.00 71 6.0 71 25 180 0 97.2 67.38470-174 1.05 75 6.0 75 10 180 0 94.4 67.88 470-176 1.05 75 3.0 75 10 18010 73.7 70.31

The following table shows the results for varying the acid chlorideconcentration, hold time and wash.

Serial Wash Hold Water, Time Acetone Area After (mL/g Percent NotebookEq. Acid Addition 004 Yield Purity Number Chloride (min) theo.) (%) (%)491-31 8.0 90 20 49.8 76.42 491-33 4.0 90 20 66.6 65.79 491-35 6.0 90 1068.2 69.04 491-37 8.0 120 10 55.2 73.58 491-47 6.0 120 20 69.3 67.16491-49 4.0 120 10 73.2 67.74 491-57 6.0 60 20 69.8 69.91 491-59 8.0 90 082.3 60.63 491-61 4.0 90 0 89.6 62.64 491-76 6.0 90 10 72.1 74.20491-106 6.0 60 0 91.9 61.13 514-53 4.0 60 10 61.0 71.20 514-67 6.0 120 051.4 70.66 514-75 6.0 90 10 36.5 79.06 514-77 6.0 90 10 37.9 79.42514-81 8.0 60 10 21.6 76.76

The following table shows the results from a coupling using sodiumhydroxide under varying conditions.

Time after EFC Time Final NaOH 016 (mol/ after acet/ Purity Notebook (%addition mol EFC water Yield (Area Number excess) (min.) 016) (min)ratio (%) %) 558-152 10 0 3 30 0.5 61 70.6 558-154 10 0 3 0 2 62 72.5558-158 0 30 3 0 2 59 73.2 558-160 0 0 2 0 2 45 74.7 558-162 10 30 2 300.5 14 74.8 558-164 10 30 3 0 0.5 59 74.2 558-170 0 30 3 30 0.5 61 69.4558-172 10 30 3 30 2 64 71.7 558-174 10 0 2 30 2 47 72.4 558-176 0 30 230 2 44 74.6 558-182 10 30 2 0 2 44 74.1 558-184 0 0 3 0 0.5 60 72.5558-186 0 30 2 0 0.5 42 75.3 582-001 0 0 3 30 2 64 70.3 582-003 0 0 2 300.5 46 73.9 582-005 10 0 2 0 0.5 44 73.9

The following table shows further results using sodium hydroxide duringcoupling reactions.

H2O:016 RXN. ratio Acetone:EFC Purity Notebook Temp. (mL/g ratio (mL/gYield (Area Number (° C.) 016) EFC) (%) %) 582-014 30 30 30 44 78.7582-016 10 30 30 57 78.8 582-018 10 5 30 40 75.1 582-020 30 30 0 42 75.2582-026 10 30 0 60 73.3 582-028 30 5 0 16 83.7 582-032 30 5 30 26 76.6

FIG. 27 is a chemical scheme showing the reaction between anaminoalkyl-diketopiperazine and EFC, followed by saponification of theethyl moiety.

Saponification procedures: Fixed pH. A 500 mL 4-neck round bottom flaskwas charged with 26.75 g of an 18.68% 016 solution (5.00 g 016 real,13.3 mmol) and 108 mL of water. Then, 15 mL of 9.5 M sodium hydroxidewas added to the reaction. A solution of 4.6 mL (33 mmol, d=1.17) EFC in125 mL of THF was added drop-wise by addition funnel, and the resultingmixture was held for 30 minutes to facilitate the coupling reaction. Thereaction mixture was then treated with 10 mL of 9.5 M sodium hydroxideand heated to reflux (67° C.) to saponify. After 1.5 hours at reflux,the reaction was cooled to 30° C. and 15 mL (−^(|)150 mmol) of −^(|)10MHCl was added. The reaction was stirred for 30 minutes. The resultingsolids were collected by filtration, washed with water (3×50 mL),methanol (3×50 mL), and acetone (3×50 mL), and dried in a vacuum oven at50° C. for a minimum of 15 hours. Weight percent purity was determinedby TM54782.

Alternatively, reagent ratios were the same as described above with thefollowing exceptions. For 2/EFC coupling, reaction was adjusted to pH 11with 9.5 M sodium hydroxide. For saponification, additional 9.5 M NaOHwas added to adjust the solution to a predetermined variable pH. Productprecipitation was conducted as described above.

Four acids (hydrochloric, sulfuric, phosphoric, and acetic) wereevaluated for 4 (R═H) precipitation. Hydrochloric acid gave the bestproduct yield and quality (FIG. 28). Back-titration of 4 (R═H)precipitated with HCl demonstrated full conversion to the di-acid.

FIG. 29 shows the results for variable conditions used in the couplingreaction of FIG. 27.

FIG. 30 shows a chemical scheme for in situ deprotection of anaminoalkyl-diketopiperazine followed by coupling with EFC.

4 (R=Et) Preparation without pH Control:

A 500 mL 3-neck round bottom flask was equipped with a magnetic stirrer,temperature readout/controller, and an addition funnel with a nitrogenhead. The exhaust gas was vented to a caustic scrubber. The flask wascharged with TFA-DKP (9.68 g, 0.022 mol) and acetone (10 mL) andstirring was initiated. Sodium hydroxide (5.18 g, 0.13 mol) dissolved inwater (25 mL) was added to the TFA-DKP slurry. An exotherm of −^(|)13°C. was observed after the sodium hydroxide addition. The mixture wasstirred at room temperature for about 10 minutes. The resulting clearyellow solution was pH 13. EFC (8.94 g, 0.055 mol) dissolved in acetone(10 mL) was added to the reaction mixture via addition funnel over 5-10minutes. During the EFC addition, the mixture pH dropped to about 4, soadditional sodium hydroxide (1.1 g, 0.028 mol) dissolved in water (10mL) was added to raise the pH to about 9. The mixture was stirred atroom temperature for about an hour, quenched with water (50 mL) and thenstirred for an additional 30 minutes. The resulting solids werecollected by filtration, washed with water (2×100 mL) and acetone (2×100mL) and dried in a vacuum oven at 50° C. overnight. The solids wereanalyzed using HPLC TM5466. Reaction yield, and 4 (R=Et) area % and wt %purity were monitored.

4 (R=Et) Preparation using pH Control:

A 500 mL 4-neck round bottom flask was equipped with a magnetic stirrer,a temperature readout/controller, a syringe pump for EFC addition, a pHprobe and a syphon tube with adaptor for 25% NaOH attached to additionand stirring was initiated. Sodium hydroxide (4.32 g, 0.11 mol)dissolved in water (60 mL) was added to the TFA-DKP slurry. An exothermof ˜14° C. was observed after the sodium hydroxide addition. The mixturewas stirred at room temperature for about 40 minutes. The resultingclear yellow solution was pH 11.9. EFC in acetone (18.38 g, 0.11 mol;prepared as above) was added to the reaction mixture via syringe pumpover 20 minutes. The solution pH was held at 8.5 by addition of 25% NaOHusing an addition pump. At the end of the EFC addition, the reaction pHwas 9.7 and the reaction temperature was 50° C. The mixture was stirredat room temperature for about 30 minutes, quenched with water (100 mL)and then stirred for an additional 30 minutes. The resulting solids werecollected by filtration, washed with water (2×100 mL) and acetone (2×100mL) and dried in a vacuum oven at 50° C. overnight. The solids wereanalyzed using HPLC TM5466. Reaction yield, volume of base consumed, and4 (R=Et) area % and wt % purity were monitored.

The coupling of deprotected TFA-DKP and EFC was conducted. When thereaction was conducted without pH control, the reaction mixture becameacidic during the EFC addition. Additional NaOH was required to raisethe pH to 7-8 and drive the reaction to completion. When the couplingreaction was conducted under pH control, crude 4 (R=Et) was obtained in80% yield and 79.9 wt % purity. A second pH-controlled reaction wasconducted, and evaluated use of neat EFC instead of an EFC/acetonesolution. Here, the reaction pH at the end of the EFC addition was 4.4,in spite of the addition of 2.32 molar equivalents of NaOH (relative toEFC). These studies demonstrated that the pH controlled direct couplingof deprotected TFA-DKP and EFC gives crude 4 (R=Et) in better yield andpurity than conditions that did not use pH controlled conditions.

The terms “a” and “an” and “the” and similar references used in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext.

Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of any and all Markush groups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.Furthermore, references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that may be employed are within the scopeof the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention may be utilized inaccordance with the teachings herein. Accordingly, the present inventionis not limited to that precisely as shown and described.

Having shown and described an embodiment of the invention, those skilledin the art will realize that many variations and modifications may bemade to affect the described invention and still be within the scope ofthe claimed invention. Additionally, many of the elements indicatedabove may be altered or replaced by different elements which willprovide the same result and fall within the spirit of the claimedinvention. It is the intention, therefore, to limit the invention onlyas indicated by the scope of the claims.

What is claimed is:
 1. A method of preparing a diketopiperazine ofFormula I (n=1-7) comprising:

mixing 4-nitrophenol, an inorganic metallic base, and ethyl fumarylchloride in an organic solvent to produce a monoethyl fumarate ester;adding an aminoalkyl-diketopiperazine; wherein the mono-ethyl fumarateester is reacted with the aminoalkyl-diketopiperazine in situ withoutpurification.
 2. The method of claim 1, wherein the organic solvent isselected from acetone, acetonitrile, ethyl acetate, tetrahydrofuran, anddichloromethane.
 3. The method of claim 1, wherein the base is selectedfrom sodium carbonate and sodium hydroxide.
 4. The method of claim 1,wherein the base is provided in an amount of 1 to 2 equivalents based onthe amount of 4-nitrophenol.
 5. The method of claim 1, wherein the ethylfumaryl chloride is provided in an amount of 0.5 to 2 equivalents basedon the amount of 4-nitrophenol.
 6. The method of claim 1, wherein theinorganic metallic base is added as an aqueous mixture to a solution of4-nitrophenol in the organic solvent.
 7. The method of claim 1, whereinthe ethyl fumaryl chloride is added to a mixture of 4-nitrophenol andthe inorganic metallic base, with cooling.
 8. The method of claim 1,wherein the aminoalkyl-diketopiperazine is added as a mixture in aninorganic solvent.
 9. The method of claim 8, wherein theaminoalkyl-diketopiperazine-containing mixture further comprises a baseselected from sodium carbonate and sodium hydroxide.
 10. The method ofclaim 8, wherein the mixture is heated after addition of theaminoalkyl-diketopiperazine-containing mixture.
 11. The method of claim1, wherein the reaction is quenched with water.
 12. A method ofpreparing a diketopiperazine of Formula I (n=1-7) comprising:

mixing mono-ethyl fumarate with a proton scavenger and at least one of:diphenylphosphoryl azide, pivaloyl chloride, chlorosulfonyl isocyanate,and trifluoroacetic anhydride in an organic solvent to form a firstreaction mixture; and adding an aminoalkyl-diketopiperazine to form asecond reaction mixture.
 13. The method of claim 12, wherein the protonscavenger is selected from triethylamine, sodium carbonate, and sodiumhydroxide.
 14. The method of claim 12, wherein sodium carbonate is addedwith the aminoalkyl-diketopiperazine.
 15. The method of claim 12,wherein the aminoalkyl-diketopiperazine is added without priorpurification of the first reaction mixture.
 16. The method of claim 12,wherein the organic solvent is selected from acetone, acetonitrile,ethyl acetate, tetrahydrofuran, and dichloromethane.
 17. The method ofclaim 12, wherein the organic solvent is tetrahydrofuran.
 18. A methodof preparing a diketopiperazine of Formula I (n=1-7) comprising:

mixing an aminoalkyl diketopiperazine of Formula II,

an inorganic metallic base, and an anhydride of mono-ethyl fumarate inan organic solvent.
 19. The method of claim 18, wherein the anhydride ofmono-ethyl fumarate is a mixed anhydride.
 20. The method of claim 18,wherein the an inorganic metallic base is sodium carbonate.